Method for chemoselection

ABSTRACT

A method of radiation-free hematopoietic stem cell (HSC) transplantation comprises administering to a mammalian subject one or two doses of 2 to 10 mg/kg body weight of a purine base analog, such as 6TG as a pre-conditioning step. The method further comprises engrafting into the subject hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient donor HSCs within 48 to 72 hours of the pre-conditioning step; and administering to the subject about 1 to 5 mg/kg of the purine base analog every two to four days for two to eight weeks following the engrafting step. The method is performed in the absence of pre-conditioning via radiation. The subject is therefore not treated with myeloablative radiation in preparation for transplantation, and thus the subject is free of myeloablative radiation-induced toxicity.

This application claims the benefit of U.S. provisional patentapplication No. 61/477,440, filed Apr. 20, 2011, the entire contents ofeach of which are incorporated herein by reference. Throughout thisapplication various publications are referenced. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to describe more fully thestate of the art to which this invention pertains.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support of Grant No. AI067769,awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND

Hematopoietic stem cell transplantation (HSCT) is a mainstay oftreatment for many hereditary disorders and lymphohematopoieticmalignancies (1). Furthermore, hematopoietic stem cells (HSC) in generalrepresent an important target for ex vivo gene therapy. Gene transferinto HSC provides a potential strategy for both correcting monogenicdefects and altering drug sensitivity of normal BM to cytotoxic drugs.These applications have significant therapeutic potential but have beenlimited by low gene transfer into HSC. Recent advances, such as improvedcytokines for minimizing commitment during ex vivo manipulation,fibronectin-assisted gene transfer and enrichment of HSC prior genetransfer have improved the efficiency of gene transfer into human cellsand enhanced human gene therapy trials (2). However, the efficiency ofgene transfer into HSC and the engraftment of large numbers oftransduced cells remains a major challenge to broaden the application ofthis technology for the successful treatment of cancer and monogeneticdiseases.

In order to enhance engraftment of gene-modified HSC and to decrease thetime needed for lymphohematopoietic reconstitution after HSCT, in vivoselection strategies employing drug resistance genes such asdihydrofolate reductase (DHFR) (3) or multiple drug-resistance gene 1(MDR1) (4, US1996/017660) have been tested but have generally failed dueto unacceptable toxicity (5) or insufficient selection efficiency (6).Currently, mutant forms of O6-methylguanine-DNA-methyltransferase (MGMT)are being tested for their ability to confer chemoprotection againstBCNU or temozolomide in combination with O6-benzylguanine (2,7,US1997/004917), but these agents pose a considerable risk of toxicity,and recent observations suggest that mutant MGMT may confer a selectivedisadvantage when expressed at high levels (8). In US2003032003AA aselection strategy has been described for selecting HPRT-deficient cellsin vivo by 6TG. However, in this patent application either irradiationis still used for preconditioning prior in vivo selection or in vivoselection is performed in cycles with recovery periods in between 6TGadministration.

Furthermore, the suggested 6TG dose is high and administrated over along time period (200 mg/kg total dose over 55 days). In addition, anapproach to inactivate HPRT expression in BM and subsequently select thedonor cells with 6TG in vivo has been reported by Porter and DiGregorias “interfering RNA mediated purine analog resistance” (‘iPAR’). Thisreport demonstrated the feasibility of HPRT inactivation in HSC with alentiviral vector expressing shRNA targeting Hprt and enrichment forthese altered hematopoietic cells with 6TG in mice in vivo. However, inthis report, pre-conditioning was still performed by total bodyirradiation, and in vivo chemoselection was not initiated until at least4 weeks post-transplant. In addition, 6TG was administered either as ashort pulse or at dosages chosen to be only moderately myelosuppressive,and it is not clear whether adequate levels of HSC transduction wereachieved by the second-generation lentiviral vectors employed in theirstudy. Overall, the engraftment levels reported were variable andrelatively modest.

There remains a need for more effective methods of HSCT that avoidtoxicity while reconstituting bone marrow cells.

REFERENCES

-   (1) Bhattacharya D, Ehrlich L I, Weissman I L. Eur J Immunol. 2008;    38:2060-2067.-   (2) Milsom M D, Williams D A. DNA Repair (Amst). 2007; 6:1210-1221.-   (3) Williams D A, Hsieh K, DeSilva A, Mulligan R C. J Exp Med. 1987;    166:210-218.-   (4) Sorrentino B P, Brandt S J, Bodine D, et al. Science. 1992;    257:99-103.-   (5) Zaboikin M, Srinivasakumar N, Schuening F. Cancer Gene Ther.    2006; 13:335-345.-   (6) Southgate T, Fairbairn L J. Expert Rev Mol Med. 2004; 6:1-24.-   (7) Neff T, Beard B C, Kiem H P. Blood. 2006; 107:1751-1760.-   (8) Schambach A, Baum C. DNA Repair (Amst). 2007; 6:1187-1196.-   (9) Porter C C, DeGregori J. Blood. 2008; 112:4466-4474.-   In vivo selection of primitive hematopoietic cells (Patent    pubication WO/1998/019540) US1996/017660-   Use of mutant alkyltransferases for gene therapy to protect from    toxicity of therapeutic alkylating agents (Patent publication    WO/1997/035477) US1997/004917-   In vivo selection (Patent publication WO/1997/043900) US2003032003AA

SUMMARY

The invention provides a method of radiation-free hematopoietic stemcell (HSC) transplantation. Typically, the method comprisesadministering to a mammalian subject one or two doses of 2 to 10 mg/kgbody weight of a purine base analog as a pre-conditioning step. Themethod further comprises engrafting into the subjecthypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient donorHSCs within 48 to 72 hours of the pre-conditioning step; andadministering to the subject about 1 to 5 mg/kg of the purine baseanalog every two to four days for two to eight weeks following theengrafting step. The method is performed in the absence ofpre-conditioning via radiation. The subject is therefore not treatedwith myeloablative radiation in preparation for transplantation, andthus the subject is free of myeloablative radiation-induced toxicity.

Representative examples of the purine base analog include: 6-thioguanine(6TG), 6-mercaptopurine (6-MP), and azathiopurine (AZA). In oneembodiment, the purine base analog is 6TG. In some embodiments, thetotal 6TG dosage administered to the subject does not exceed 105 mg;typically, the total 6TG dosage administered to the subject does notexceed 75 mg. In one embodiment, the administering of purine base analogis performed every 3 days and for not more than four weeks following theengrafting step.

Subjects treated in accordance with the method will exhibit over 75%genetically modified hematopoietic cells. In some embodiments, thesubject exhibits over 95% genetically modified hematopoietic cells.

The HPRT-deficient HSCs to be transplanted can be renderedHPRT-deficient using conventional methods known to those skilled in theart. Representative methods include, but are not limited to,introduction of sequences encoding zinc finger nucleases (ZFNs),transcriptional activator-like effector nucleases (TALENs), smallfragment homologous recombination (SFHR) template strands, inhibitoryRNAs (siRNAs) or microRNAs (miRNAs), antisense RNAs, trans-splicingRNAs, ribozymes, intracellular antibodies, or dominant-negative orcompetitive inhibitor proteins. The transplanted HSCs can be autologous,syngeneic, or allogeneic.

In some embodiments, the HPRT-deficient HSCs to be transplanted havebeen genetically modified. The subject may have a hereditary or geneticdisorder, an acquired disease affecting lymphohematopoietic cells, suchas human immunodeficiency virus (HIV) infection or acquired immunedeficiency syndrome (AIDS), or a lymphohematopoietic malignancy. Thegenetic modification of the donor HSCs can extend beyond rendering thecells HPRT-deficient, and also serve to treat or correct a condition.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Optimization of 6TG conditioning regimen. (FIG. 1A) As adose-finding study, HPRT-wt mice were injected i.p. with vehiclecontrol, or varying 6TG doses ranging from 2.5 to 10 mg/kg, asindicated, on Day 1 (n=3 per group), or with two doses of 10 mg/kg onDay 1 and Day 3 (n=3), respectively. On Day 4 after the first 6TG dose,paraformaldehyde-fixed bone sections were stained with H&E, and BMhistology was examined. Representative photomicrographs (40×magnification) are shown for each 6TG conditioning regimen. (FIG. 1B)Representative photomicrographs showing overall and detailed BMhistology at low (10×) and high (100×) magnification from HPRT-wt micetreated with control vehicle, or with the optimized conditioning regimenconsisting of two doses of 10 mg/kg 6TG on Day 1 and Day 3. Histologicalanalyses were performed on Day 4, as above.

FIGS. 2A-2B. Lack of progressive myelotoxicity after injection withconditioning doses of 6TG in HPRT-deficient mice, and low engraftmentrate with 6TG conditioning alone. (FIG. 2A) HPRT-wt and HPRT-deficientmice were injected i.p. with 10 mg/kg 6TG on Days 1 and 3. On Day 7after the first 6TG dose, paraformaldehyde-fixed bone sections werestained with H&E, and BM histology was examined. Representativephotomicrographs (40× magnification) are shown. (FIG. 2B) Treatmentschedule: HPRT-wt female recipient mice (n=4) received the firstconditioning dose of 6TG (10 mg/kg, i.p.) on Day −2, then weretransplanted with HPRT-deficient male BM, followed by a secondconditioning dose of 6TG (10 mg/kg, i.p.) on Day 0. BM was analyzed onDay 7 after the first 6TG dose. Paraformaldehyde-fixed bone sectionsstained with H&E (40× magnification) are shown.

FIG. 3. Dose-response and time course of chronic low-dose 6TGmyelotoxicity in HPRT-wt vs. HPRT-deficient mice. HPRT-wild type miceand HPRT-deficient mice were treated every 3 days with different dosagesof 6TG or vehicle control (n=3 per group), as shown above each panel.For HPRT-wild type mice, histology was examined at the following timepoints up to 60 days after initiation of treatment: Vehicle control (Day60), 6TG 0.25 mg/kg (Day 60), 6TG 0.5 mg/kg (Day 60), 6TG 1.0 mg/kg (Day38), 6TG 2.5 mg/kg (Day 28), 6TG 5.0 mg/kg (Day 22). For HPRT-deficientmice, histology was examined on Day 60 for all animals. Representativephotomicrographs of paraformaldehyde-fixed bone sections stained withH&E (40× original magnification) are shown.

FIGS. 4A-4B. Optimization of combined 6TG conditioning and in vivochemoselection strategy. Treatment schedule: HPRT-wt female recipientmice received the first conditioning dose of 6TG (10 mg/kg, i.p.) on Day−2, then were transplanted with HPRT-deficient male BM along with asecond conditioning dose of 6TG (10 mg/kg, i.p.) on Day 0. In vivochemoselection was then performed with repeated i.p. injections of 2.5mg/kg or 5.0 mg/kg 6TG every 3 days for a period of 2 weeks (FIG. 4A) or4 weeks (FIG. 4B), as indicated. Representative photomicrographs of bonemarrow from paraformaldehyde-fixed sections stained with H&E (40×original magnification) are shown.

FIG. 5. Long-term hematopoietic reconstitution after transplantation ofHPRT-wt recipients with HPRT-deficient donor-derived BM using combined6TG conditioning and chemoselection. Bar graphs show the percentage ofdonor-derived cells in bone marrow (BM) and peripheral blood leukocytes(PBL) as determined by XY chromosome FISH analysis at 4 weeks (i.e.,immediately after the end of the chemoselection period), and at 4months, 7 months, and 12 months after transplantation, as indicated.

DETAILED DESCRIPTION

The invention provides a novel in vivo chemoselection strategy thatexploits the essential role of hypoxanthine-guaninephosphoribosyltransferase (HPRT)-mediated conversion of 6-thioguanine(6TG) to thioguanine nucleotide in 6TG myelotoxicity. BecauseHPRT-deficiency per se does not impair hematopoietic cell development orfunction, it can be removed from hematopoietic cells to be used fortransplantation. The in vivo chemoselection strategy involves HSCTperformed with HPRT-deficient donor HSCs using 6TG both formyeloablative conditioning of HPRT-wild type recipients and for a singlecycle in vivo chemoselection process of donor cells. The invention isbased on the development and discovery of a dosing schedule at which 6TGinduces selective myeloablation without any adverse effects onextra-hematopoietic tissues, while engrafted HSC deficient in HPRTactivity are highly resistant to the cytotoxic effects of 6TG. With thisstrategy of combined 6TG conditioning and chemoselection, efficient andhigh engraftment of HPRT-deficient donor HSC with low overall toxicitycan be achieved. 6TG in vivo chemoselection allows long-termreconstitution of immunophenotypically normal bone marrow (BM) byamplifying the self-renewing, pluripotent HSC population fromHPRT-deficient donor (or engrafted) BM.

The method described herein for highly efficient and overall non-toxicconditioning and single cycle in vivo chemoselection is generallyapplicable as a strategy to improve HSCT engraftment efficiency andtransplantation outcome, and to confer a selective advantage togenetically modified cells after ex vivo gene therapy. The in vivochemoselection strategy exclusively employs 6TG, or other purine baseanalog, for both pre-conditioning and single cycle chemoselection ofHPRT-deficient donor HSC, and is capable of achieving highly efficientengraftment and long-term reconstitution, with replacement of >95% ofthe recipient BM. This strategy is applicable for improving theengraftment of large numbers of ex vivo manipulated HSC to broaden theapplication of gene therapy in general.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “radiation-free hematopoietic stem cell (HSC)transplantation” means that the recipient is not subjected tomyeloablative conditioning via radiation. Instead, conditioning isachieved through administration of 6TG, typically administered duringthe 48 hours prior to (and including the day of) engraftment with donorHSCs.

As used herein, “HPRT-deficient” includes both cells that are naturallyHPRT-deficient, and those rendered HPRT-deficient through geneticmodification.

As used herein, “donor HSCs” or “donor cells” refers to the cells to beengrafted, regardless of whether the HSCs are derived from the recipientof the transplant or another subject. Thus, cells harvested from asubject can be modified and engrafted back into the same subject,becoming “donor cells”. These may be referred to herein as “donorcells”, “engrafted cells” or “transplanted cells”.

As used herein, “a” or “an” means at least one, unless clearly indicatedotherwise.

Combined Conditioning and Chemoselection for HematopoieticTransplantation

The invention provides a method of radiation-free hematopoietic stemcell (HSC) transplantation. Typically, the method comprisesadministering to a mammalian subject one or two doses of 2 to 10 mg/kgbody weight of a purine base analog as a pre-conditioning step. Themethod further comprises engrafting into the subjecthypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient donorHSCs within 48 to 72 hours of the pre-conditioning step; andadministering to the subject about 1 to 5 mg/kg of the purine baseanalog every two to four days for two to eight weeks following theengrafting step.

The method is performed in the absence of pre-conditioning viaradiation. The subject is therefore not treated with myeloablativeradiation in preparation for transplantation, and thus the subject isfree of myeloablative radiation-induced toxicity. The method iscontemplated for use with a variety of subjects, including subjects whohave never received radiation treatment of any kind, subjects who havenever received myeloablative radiation treatment, and subjects who havereceived myeloablative treatment in the past, but not within a timeframe and/or at a dose that would be pre-conditioning for the methoddescribed herein. For example, the subject typically would not havereceived myeloablative radiation within 2 weeks, or even within 8 weeks,of the combined conditioning and chemoselection method described herein.

Representative examples of the purine base analog include: 6-thioguanine(6TG), 6-mercaptopurine (6-MP), and azathiopurine (AZA). In oneembodiment, the purine base analog is 6TG. In some embodiments, thetotal 6TG dosage administered to the subject does not exceed 105 mg;typically, the total 6TG dosage administered to the subject does notexceed 75 mg. In one embodiment, the administering of purine base analogis performed every 3 days and for not more than four weeks following theengrafting step.

Where an alternative to 6TG is used as the purine base analog, knownonline (e.g., rxlist.com) and other resources are available to guide theskilled clinician in identifying a suitable dose for use in the methodof the invention. For example, the usual oral dose for 6TG single-agentchemotherapy in pediatric patients and adults is 2 mg/kg of body weightper day; if no treatment response is observed after 4 weeks, the dosecan be increased to 3 mg/kg. As much as 35 mg/kg has been given in asingle oral dose with reversible myelosuppression observed.

For acute lymphatic leukemia, the usual initial dosage for pediatricpatients and adults is 2.5 mg/kg 6-MP of body weight per day (100 to 200mg in the average adult and 50 mg in an average 5-year-old child).Pediatric patients with acute leukemia have tolerated this dose withoutdifficulty in most cases; it may be continued daily for several weeks ormore in some patients. If, after 4 weeks at this dosage, there is noclinical improvement and no definite evidence of leukocyte or plateletdepression, the dosage may be increased up to 5 mg/kg daily. A dosage of2.5 mg/kg/day may result in a rapid fall in leukocyte count within 1 to2 weeks in some adults with acute lymphatic leukemia and high totalleukocyte counts. Once a complete hematologic remission is obtained,maintenance therapy is considered essential. Maintenance doses will varyfrom patient to patient. The usual daily maintenance dose of 6-MP is 1.5to 2.5 mg/kg/day as a single dose.

Dosage for 6-TG and 6-MP is somewhat comparable, while dosage for AZA ismore difficult to compare, because it needs first to be bioactivated to6-MP, and it is usually not used for treating leukemia. For patientsreceiving solid organ transplantation, the dose of AZA required toprevent rejection and minimize toxicity will vary with individualpatients, necessitating careful management. The initial dose is usually3 to 5 mg/kg daily, beginning at the time of transplant. AZA is usuallygiven as a single daily dose on the day of, and in a minority of cases 1to 3 days before, transplantation. Dose reduction to maintenance levelsof 1 to 3 mg/kg daily is usually possible. The dose of AZA should not beincreased to toxic levels because of threatened rejection.

The HPRT-deficient donor HSCs can be naturally HPRT-deficient, orrendered HPRT-deficient through genetic modification. In this context,“donor HSCs” refers to the cells to be engrafted, regardless of whetherthe HSCs are derived from the recipient of the transplant or anothersubject. The transplanted HSCs can be autologous, syngeneic, orallogeneic.

The genetic modification can be achieved using any of a variety of meansknown to those skilled in the art. Examples of suitable means of geneticmodification include, but are not limited to, introduction of sequencesencoding zinc finger nucleases (ZFNs), transcriptional activator-likeeffector nucleases (TALENs), small fragment homologous recombination(SFHR) template strands, inhibitory RNAs (siRNAs) or microRNAs (miRNAs),antisense RNAs, trans-splicing RNAs, ribozymes, intracellularantibodies, or dominant-negative or competitive inhibitor proteins. Themodification can be employed directly with the donor HSCs or withprogenitors. These technologies can be used for genetic modification ofa variety of cell types, including but not limited to hematopoieticprogenitor cells or hematopoietic stem cells directly, as well as othertypes of adult or embryonic stem cells or induced pluripotent stem cellsthat can be differentiated or trans-differentiated into hematopoieticprogenitor or hematopoietic stem cells.

In some embodiments, the HPRT-deficient HSCs to be transplanted havebeen genetically modified to suit a particular therapeutic objective.For example, the donor HSCs can be modified to correct a hereditarygenetic defect, to alter drug sensitivity of normal bone marrow tocytotoxic drugs, to confer resistance to infectious microorganisms thataffect lymphohematopoietic cells, to replace or re-set the endogenousimmune system, or to combat lymphohematopoietic malignancies throughreplacement of endogenous bone marrow and induction of agraft-vs.-leukemia/lymphoma effect.

More specifically, hereditary genetic defects can include, but are notlimited to, disorders of hematopoiesis including hemoglobinopathies suchas sickle cell anemia, thalassemia, hereditary spherocytosis, G6PDdeficiency, etc., disorders of immunologic or antimicrobial functionsuch as severe combined immunodeficiency (SCID), chronic granulomatousdisease (CGD), disorders of thrombopoiesis leading to coagulationdefects such as Wiscott-Aldrich syndrome (WAS), as well as other geneticstructural or metabolic disorders which can be ameliorated by geneticengineering of hematopoietic cells that travel to sites of tissuedamage, such as various forms of epidermolysis bullosa (EB), andmucopolysaccharidosis.

Diseases in which modification of the drug sensitivity of bone marrow tochemotoxic drugs would be advantageous include, but are not limited to,malignant diseases that are treated by chemotherapy agents whose maximumtolerated dosage is limited by myelotoxicity. These include lung cancer,colorectal cancer, breast cancer, prostate cancer, pancreatic cancer,gastric cancer, liver cancer, head and neck cancer, renal cellcarcinoma, bladder cancer, cervical cancer, ovarian cancer, skin cancer,sarcomas, and glioma.

Diseases in which bone marrow or hematopoietic stem cell transplantationis used to replace or reset the endogenous immune system include, butare not limited to, inflammatory bowel disease, scleroderma, and lupuserythematosis.

Diseases in which conferring resistance to infectious microorganismswould be advantageous include, but are not limited to, HIV infection andAIDS, HTLV infection, and parvovirus B19 infection.

Malignant or pre-malignant diseases of lymphohematopoiesis that aretreated by bone marrow or hematopoietic stem cell transplantationinclude, but are not limited to, acute myelogenous leukemia, acutelymphocytic leukemia, lymphoma, and myelodysplastic syndromes.

Another example of the therapeutic application of this technology wouldbe to improve the outcome of bone marrow or hematopoietic stem celltransplantation after acquired injury to endogenous lymphohematopoiesiscaused by radiation injury, and chemotoxins.

A non-therapeutic but commercially useful application of this technologywould be its use to generate humanized animal models, in which theirendogenous lymphohematopoiesis is almost entirely replaced by cells froma human donor. Once generated, such animals could be used, for example,to test the myelotoxicity of new drugs being considered for applicationto human disease. This is advantageous because the sensitivity ofhematopoiesis to various drugs can be different depending on the speciesof animal, therefore it is most desirable to test such drugs in ahumanized animal model.

Typically, the subject is a mammal. The mammalian subject can be murine,canine, feline, bovine, equine, ovine, primate or human. In oneembodiment, the subject is human.

Administration and Dosage

The compositions are administered in any suitable manner, often withpharmaceutically acceptable carriers. Suitable methods of administeringtreatment in the context of the present invention to a subject areavailable, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

The dose administered to a patient, in the context of the presentinvention, should be sufficient to effect a beneficial therapeuticresponse in the patient over time, or to inhibit disease progression.Thus, the composition is administered to a subject in an amountsufficient to elicit an effective response and/or to alleviate, reduce,cure or at least partially arrest symptoms and/or complications from thedisease. An amount adequate to accomplish this is defined as a“therapeutically effective dose.”

Routes and frequency of administration of the therapeutic compositionsdisclosed herein, as well as dosage, will vary from individual toindividual as well as with the selected drug, and may be readilyestablished using standard techniques. In general, the pharmaceuticalcompositions may be administered, by injection (e.g., intracutaneous,intratumoral, intramuscular, intravenous or subcutaneous), intranasally(e.g., by aspiration) or orally. Alternate protocols may be appropriatefor individual patients.

As is understood by those skilled in the art, doses can be convertedfrom mg/kg body weight to mg/body surface area, the latter beingsuitable for use with larger mammalian subjects, including humans.Calculators for allometric scaling are known in the art and readilyobtained online. Generally, allometric scaling uses an exponent of0.75-0.80. For more information, see West & Brown, J Exp Bio 208,1575-1592, 2005. In addition, the United States Food and DrugAdministration publishes “Guidance for Industry: Estimating the MaximumSafe Starting Dose in Initial Clinical Trials for Therapeutics in AdultHealthy Volunteers,” which is available from: Office of Training andCommunications Division of Drug Information, HFD-240 Center for DrugEvaluation and Research Food and Drug Administration 5600 Fishers LaneRockville, Md. 20857.

For example, 5 mg/kg 6TG corresponds to a dose of 15.08 mg/m2 for a 20 gmouse. This equals 0.4 mg/kg for a 65 kg human. Absorption after oral6TG administration is estimated to be 30%, therefore this i.p. dose inmice corresponds to an absorbed dose after oral administration of about1.3 mg/kg in humans. The conventional oral dose for 6TG single-agentchemotherapy in pediatric patients and adults is 2 mg/kg of body weightper day; if no treatment response is observed after 4 weeks, the dosecan be increased to 3 mg/kg.

The method of the invention provides the unexpected advantage ofavoiding toxicity subsequent to either excessive irradiation of thesubject or excessive 6TG dosage. Surprisingly, effective conditioningand reconstitution of bone marrow can be achieved using less than 105 mgtotal 6TG dosage over the course of treatment, and over a time course oftwo to eight weeks. Effective engraftment has been observed with total6TG dosages of less than 65 mg and in as few as two weeks. In addition,the claimed method allows for the option of monitoring toxicity in anindividual subject and adjusting the dosing to optimize effectiveengraftment with minimal toxicity for each subject. In some embodiments,the subject is administered 1 or 2.5 mg/kg body weight 6TG during thepost-engraftment treatments.

Subjects treated in accordance with the method will exhibit over 75%genetically modified hematopoietic cells. In some embodiments, thesubject exhibits over 95% genetically modified hematopoietic cells.Successful engraftment can be confirmed in a subject by sampling of theperipheral blood or bone marrow at various intervals aftertransplantation and chemoselection. The peripheral blood mononuclearcells can be examined by monitoring the levels of HPRT gene disruption,knockdown, or reduction in functional activity, using various standardtechniques that are generally familiar to one of ordinary skill in theart, including but not limited to polymerase chain reaction (PCR),quantitative real-time PCR (Q-PCR), surveyor nuclease assay (alsoreferred to as ‘Cel-I assay’), Southern blot analysis, Westernblot/immunoblot analysis, immunohistochemistry or immunocytochemistry,flow cytometric analysis with intracellular staining, HPRT enzymaticactivity analysis, HPLC, mass spectrometry, and the like.

EXAMPLE

The following example is presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1: Combined Preconditioning and In Vivo Chemoselection with6-Thioguanine Alone Achieves Highly Efficient Reconstitution of NormalHematopoiesis with HPRT-Deficient Bone Marrow

Purine analogs such as 6-thioguanine (6TG) cause myelotoxicity uponconversion into nucleotides by hypoxanthine-guaninephosphoribosyltransferase (HPRT). This example shows the development ofa novel and highly efficient strategy employing 6TG as a single agentfor both conditioning and in vivo chemoselection of HPRT-deficient HSC.The dose-response and time course of 6TG myelotoxicity were firstcompared in HPRT-wild type mice and HPRT-deficient transgenic mice.Dosage and schedule parameters were optimized to employ 6TG formyelo-suppressive conditioning, immediately followed by in vivochemoselection of HPRT-deficient transgenic donor bone marrow (BM)transplanted into syngeneic HPRT-wild type recipients.

At appropriate doses, 6TG induced selective myelotoxicity without anyadverse effects on extra-hematopoietic tissues in HPRT-wild type mice,while HSC deficient in HPRT activity were highly resistant to itscytotoxic effects. Combined 6TG conditioning and post transplantchemoselection consistently achieved ˜95% engraftment of HPRT-deficientdonor BM, with low overall toxicity. Long-term reconstitution ofimmunophenotypically normal BM was achieved in both primary andsecondary recipients. These results provide proof-of-concept thatsingle-agent 6TG can be used both for myelo-suppressive conditioningwithout requiring irradiation, and for in vivo chemoselection ofHPRT-deficient donor cells. The results show that by applying themyelosuppressive effects of 6TG both before (as conditioning) and aftertransplantation (as chemoselection), highly efficient engraftment ofHPRT-deficient hematopoietic stem cells can be achieved.

Clinical efficacy of ex vivo gene therapy using hematopoietic stem cellsremains dependent on imparting a selective advantage to the transplantedcells [1, 2]. In order to enhance engraftment and decrease the timeneeded for lymphohematopoietic reconstitution, in vivo selectionstrategies employing drug resistance genes such as dihydrofolatereductase (DHFR) [3] or multiple drug-resistance gene 1 (MDR1) [4, 5]have been tested, but have generally failed due to unacceptable toxicity[6] or insufficient selection efficiency [7]. Currently, mutant forms ofO⁶-methylguanine-DNA-methyltransferase (MGMT) are being tested for theirability to confer chemoprotection against BCNU or temozolomide incombination with O⁶-benzylguanine [8, 9], but these agents also pose aconsiderable risk of toxicity, and recent observations suggest thatmutant MGMT may confer a selective disadvantage when expressed at highlevels [10].

Notably, these approaches have generally relied upon transplantation ofhematopoietic progenitors over-expressing an exogenous drug resistancegene into recipients preconditioned with myeloablative irradiation;however, chemo-resistance may also be conferred by reduced levels ofendogenous enzymes that are normally essential for activation ofcytotoxic drugs. In this context, we have previously observed that highlevels of the purine nucleotide salvage pathway enzymehypoxanthine-guanine phosphoribosyltransferase (HPRT) cause increasedsensitivity to the purine analog 6-thioguanine (6TG) [11]. The firststep of the metabolic conversion of 6TG is catalyzed by HPRT [12], whichmediates the addition of ribose 5-phosphate to generate thioguanosinemonophosphate (TGMP). Thus, 6TG cytotoxicity is essentially predicatedon its HPRT-mediated conversion to thio-dGTPs, which are thenincorporated into DNA, inducing futile mismatch repair and subsequentapoptosis.

To confer myeloprotection through reduced activity, the endogenousdrug-activating enzyme should normally be highly expressed inhematopoietic progenitors, yet non-essential for normal hematopoiesis.In fact, hematopoietic progenitors normally express high levels of HPRT[13-16], which makes them extremely sensitive to 6TG. Indeed, purineanalogs such as 6-mercaptopurine (6MP), azathiopurine (Aza), and 6TGhave been used clinically for the treatment of leukemia, particularly inpediatric patients, for half a century [17], as well as forimmunosuppression in organ transplant patients, and more recently forautoimmune diseases. At higher doses, myelotoxicity is the most frequentand consistent adverse effect of 6TG when used clinically, and whenadministered at appropriate concentrations for short periods of time,6TG is strongly myelosuppressive with little toxicity to other tissuesin normal HPRT-wild type animals [11].

In contrast, bone marrow (BM) from HPRT-deficient animals is highlyresistant to 6TG [11]. Notably, however, we [11] and others [18] havefound that hematopoiesis is phenotypically and functionally normal inHprt-knockout animals, and although cases of megaloblastic anemia havebeen associated with hereditary HPRT deficiency (Lesch-Nyhan syndrome)in humans [19], this has been reported to respond well to oraladministration of adenine [20]. Furthermore, HPRT deficiency does notappear to be associated with any gross impairment of the immune systemin humans or in animals [21].

These observations suggest that HPRT-deficient hematopoietic progenitorcells, which are 6TG-resistant but otherwise normal, should have aselective advantage when transplanted into HPRT-wild type recipientsundergoing 6TG treatment, and that this strategy can be used to improvethe outcome of ex vivo gene therapy. In fact, Porter and DeGregori [22]previously demonstrated the feasibility of transducing HSC with alentiviral vector expressing Hprt-targeted shRNA and enriching theseengineered hematopoietic cells in vivo by 6TG chemoselection in mice.However, in this previous report, 6TG was employed either at dosageschosen to be only moderately myelosuppressive or only over short periodsof selection that were initiated 4 weeks or more followingtransplantation, and despite preconditioning by total body irradiation,engraftment results were relatively modest and highly variable, rangingfrom 5-50% [22].

We have now systematically examined the effects of modifying the dose,timing, and duration of 6TG administration on engraftment andhematopoietic reconstitution after transplantation of HPRT-deficientbone marrow. In order to exclude vector transduction efficiency as avariable, we employed BM from Hprt-knockout animals as ‘ideal’ donorcells, thereby enabling us to focus on the effects of modifying 6TGdosage and scheduling parameters both (i) pre-transplantation formyelosuppressive conditioning of HPRT-wild type recipients, and (ii)post-transplantation for chemoselective amplification of HPRT-deficientdonor cell populations. Consequently, here we describe the developmentof a novel regimen that sequentially employs 6TG as a single agent forboth preconditioning and in vivo chemoselection, and show that thiscombination regimen rapidly and consistently achieves highly efficientengraftment and long-term reconstitution.

Materials and Methods

Mice

Hprt-deficient B6.129P2-Hprt1^(b-m3)/J (CD45.2) mice, Hprt-wild type(wt) C57BL/6J, and B6.SJL-Ptprc^(a)Pepc^(b)/BoyJ (CD45.1) mice wereoriginally obtained from the Jackson Laboratory (Bar Harbor, Me.).B6.129P2-Hprt1^(b-m3)/J mice carry a 55 kb deletion spanning thepromoter and the first 2 exons of the Hprt-gene [23]. Mice were bred andmaintained in the institutional specific-pathogen-free animal facilityunder standard conditions according to institutional guidelines.

6TG Treatment

C57BL/6J and B6.129P2-Hprt1^(b-m3)/J mice were injectedintraperitoneally (i.p.) with 200 μl of varying doses of 6TG(Sigma-Aldrich, Saint Louis, Mo.) at different time points as describedin the figure legends. Control animals were i.p. injected with 200 μl ofsterile H₂O.

Bone Marrow Transplantation and 6TG In Vivo Chemoselection

Female recipient C57BL/6J (HPRT-wt) or B6.SJL-Ptprc^(a)Pepc^(b)/BoyJ(HPRT-wt, CD45.1) mice were treated with 10 mg/kg 6TG by i.p. injection48 hrs before HSCT. For HSCT, 0.8-1×10⁷ nucleated BM cells isolated fromB6.129P2-Hprt1^(b-m3)/J male mice (CD45.2) were intravenously injectedinto HPRT-wt recipients. 6TG (10 mg/kg) was again administered by i.p.injection 2 hrs later, and every 3 days thereafter, at 5 mg/kg for 4weeks. Serial transplantation into secondary recipient mice wasperformed using the same cell dose and 6TGpreconditioning/chemoselection regimen as above, but using BM fromprimary recipient mice that had undergone transplantation with 6TG invivo chemoselection 6 months previously.

Mouse Chromosome X- and Y-Specific Fluorescence In Situ Hybridization

FISH was performed on BM and PBL cells using the mouse-specific WholeChromosome-Y paint probe/RAB9 (XqF1) DNA probes mix (Kreatech,Amsterdam, Netherlands) according to the manufacturer's protocol. Todetermine the percentage of male donor HPRT-wt cells in the femalerecipient C57BL/6J mice, 200 nuclei per slide were counted using afluorescence microscope (Zeiss) equipped with appropriate dual andtriple-colour filters. The following criteria were applied for analyzingFISH: (1) quality of the nuclei was evaluated via DAPI staining, (2)green fluorescent signal for the Y-chromosome was scored, (3) redfluorescent signal for X-chromosome was scored, (4) absence of the greenfluorescent signal for the Y-chromosome nucleus was recorded as female,even when only one X-chromosome was detectable (Supplementary FIG. 51;see Experimental Hematology 2012, 40:3-13 for supplementary figures).

Immunophenotypic Analysis of Hematopoietic Tissues

After blocking with Mouse BD Fc Block (BD Biosciences, San Jose,Calif.), BM, PBL, thymus, or spleen cells were stained with FITC-, PE-,PerCP- or APC-conjugated rat anti-mouse antibodies against CD45, CD45.2,CD4, CD8, Mac1/Gr1, B220, Sca-1, c-kit, and lineage antibody cocktail.Antibodies were received from Biolegend (San Diego, Calif.) or BDBiosciences. Flow cytometric data were acquired on a BD LSRII running BDFACSDiva (BD Biosciences) and analyzed using FlowJo software (TreeStar,Ashland, Oreg.) (Supplementary Figure S2).

Histopathological Analysis

Necropsy of all mice used in this study and histological examination ofall thoracic and abdominal organs and bone marrow was performed by theUCLA Division of Laboratory Animal Medicine Diagnostic ServiceLaboratory. Tissues were routinely processed or decalcified ifnecessary, and paraffin sections were cut and stained with hematoxylinand eosin (H&E).

Statistical Analysis

Data were analyzed using the QuickCalcs statistical software program(Graphpad Software Inc.). Unpaired t-tests were used to calculate pvalues, and p<0.05 was considered statistically significant.

Results

Acute Myelotoxicity of 6TG in HPRT-Wt Mice

The high levels of HPRT expression in hematopoietic progenitors arepredicted to mediate a selective myelotoxic effect of 6TG and enable itsuse as a conditioning regimen. Accordingly, we examined the short-termeffects on BM after bolus injections of 6TG at various dosages inHPRT-wt mice. Intraperitoneal injection of a single dose of 2.5 mg/kg,5.0 mg/kg, or 10 mg/kg 6TG was performed on Day 1, or injection of twodoses at 10 mg/kg on Days 1 and 3, and BM histology was examined on Day4. Increasing myelotoxicity was observed as the total 6TG dosage wasincreased (FIG. 1A). Vascular structures became progressively prominentand cells of both erythroid and myeloid lineages were depleted. Onlyvascular endothelium, mesenchymal cells, some mature granulocytes andmacrophages, and occasional hematopoietic progenitor cells remained atthe highest 6TG dose tested (FIG. 1B).

No readily apparent clinical signs were observed on Day 4 with any ofthe above regimens, but when the observation period was extended, weightloss and pallor of the extremities was seen by Day 7. Histologicalexamination of BM showed severity increasing between Day 4 and Day 7even without administration of additional doses of 6TG (FIG. 1A, 2A). Inconcordance with the clinical and histological findings, BM of HPRT-wtmice treated with two doses of 10 mg/kg 6TG and analyzed on Day 7 showeda significant quantitative decrease in the BM nucleated cell countrecovered from one femur and tibia (4.7×10⁶±1.0×10⁶, n=3) compared tothat of vehicle control-treated HPRT-wt mice (1.1×10⁷±0.2×10⁷, n=3)(p<0.01).

Immunophenotypic analysis of the remaining BM hematopoietic cells byflow cytometry (Table 1) revealed that the relative proportion of KLS(lin⁻/c−kit⁺/sca-1⁺) progenitors, which includes HSC with long-termmultilineage reconstitution activity [24], significantly decreased3-fold by Day 4 (p<0.01), and 10-fold by Day 7 (p<0.001) (Table 1-iii,-iv). The relative percentage of mature CD8+ and CD4+ T cellsprogressively increased over time after 6TG administration, reaching upto 7-fold compared to the controls by Day 7 (p<0.001), likely due to themassively dilated blood vessels and influx of peripheralblood/hemorrhage, as can be also seen in the BM histology. The relativepercentage of B220+ cells showed no significant change by Day 4, but haddoubled compared to controls by Day 7 after 6TG administration.

TABLE 1 Immunophenotypic analysis of BM hematopoietic cells after 6TGconditioning regimen. BM cells were stained with the following ratanti-mouse antibodies: CD45-FITC, CD4-PE, CD8-APC, B220-PerCP,Mac1/Gr1-PE, Sca-1-PE, and c-kit-FITC, and examined by flow cytometry.Treatment and analysis schedules are indicated by small roman numerals,and are the same as in FIG. 1. HPRT- Cell HPRT-wild type deficientpopulation i ii iii iv v CD45⁺ 91.5 ± 2.4 95.7 ± 0.8 95.7 ± 0.1 82.8 ±5.1 90.0 ± 2.0 CD4⁺  3.5 ± 0.4 11.9 ± 2.5  9.6 ± 0.6 20.5 ± 3.1  2.9 ±1.7 CD8⁺  3.6 ± 0.1  5.3 ± 1.7  5.2 ± 0.4 25.0 ± 2.7  2.7 ± 0.4 B220⁺28.4 ± 3.3 23.1 ± 2.2 26.2 ± 1.8 56.3 ± 1.5 10.7 ± 3.5 Mac1⁺/Gr1⁺ 75.8 ±1.1 85.4 ± 1.8 81.0 ± 1.3 61.8 ± 3.0 79.3 ± 2.9 KLS (HSC)  4.2 ± 0.6 1.3 ± 0.2  1.4 ± 0.5  0.4 ± 0.2  2.0 ± 0.6 (i): Vehicle control on Day1, analysis on Day 4. (ii) Single dose of 10 mg/kg 6TG on Day 1,analysis on Day 4. (iii): Two doses of 10 mg/kg 6TG on Days 1 and 3,analysis on Day 4. (iv) & (v): Two doses of 6TG 10 mg/kg on Days 1 and3, analysis on Day 7. The last two columns show the results of the same6TG dosage and schedule (10 mg/kg 6TG × 2 doses) in HPRT-wt (iv) andHPRT-deficient (v) mice, respectively. Percentages of the indicatedhematopoietic cell subpopulations are expressed as mean % ± SD of totalCD45+ cells (n = 3 per group).

Notably, liver enzymes were not elevated following this conditioningregimen in HPRT-wt mice, indicating the selectivity of 6TG cytotoxicityfor hematopoietic progenitors at this dosage, and suggesting itspotential for use as a myelosuppressive conditioning regimen.

Lack of Myelotoxicity at 6TG Conditioning Dosage in HPRT-Deficient Mice

In contrast to the above findings, when HPRT-deficient mice were treatedwith the maximum dosing (two doses of 10 mg/kg 6TG on Days 1 and 3),bone marrow histology remained completely unaffected at day 7 (FIG. 2A),and was comparable to that of the vehicle control group (FIG. 1A).Importantly, the overall count of nucleated BM cells obtained from onefemur and tibia of 6TG-treated HPRT-deficient mice (1.5×10⁷±0.3×10⁷,n=3) was also comparable to that of vehicle control-treated HPRT-wt mice(1.1×10⁷±0.2×10⁷, n=3), and was significantly higher than in HPRT-wtmice treated with the same 6TG regimen (4.7×10⁶±1.0×10⁶, n=3) (p<0.005).

Furthermore, the ratios of hematopoietic cell subpopulations in the BMof the 6TG-treated HPRT-deficient mice (Table 1-v) were comparable tothose of vehicle control-treated HPRT-wt mice (Table 1-i) and untreatedHPRT-wt mice (Table 2, BM % column), as well as untreated HPRT-deficientmice (Table 3, BM % column).

TABLE 2 Immunophenotypic analysis of hematopoietic cells intreatment-naïve HPRT-wt mice. BM, PBL, spleen (S) and thymus (T) werestained with the following rat anti-mouse antibodies: CD45- FITC (BM,PBL, T, S), CD4-PE (BM, PBL, T, S), CD8-APC (BM, PBL, T, S), Mac1/Gr1-PE(BM, PBL, S), B220-PerCP (BM, PBL, S), Sca-1-PE (BM), and c-kit-FITC(BM), and examined by flow cytometry. Percentages of the indicatedhematopoietic cell subpopulations are expressed as mean % ± SD of totalCD45+ cells (n = 3 per group). Cell population BM (%) PBL (%) Thymus (%)Spleen (%) CD45⁺ 92.6 ± 3.2 98.6 ± 0.5 98.7 ± 0.6 97.1 ± 1.0 CD4⁺  3.9 ±1.6 12.2 ± 2.6  6.5 ± 2.1 19.7 ± 1.7 CD8⁺  1.6 ± 0.8 11.4 ± 1.7  3.6 ±1.2 16.5 ± 0.3 CD4⁺/CD8⁺ 85.6 ± 4.3 B220⁺ 20.4 ± 8.2  47.8 ± 13.2 54.4 ±4.9 Mac1⁺/Gr1⁺ 87.3 ± 8.1 36.1 ± 5.0 18.7 ± 3.9 KLS (HSC)  3.6 ± 2.4

TABLE 3 Immunophenotypic analysis of hematopoietic cells intreatment-naïve HPRT-deficient mice. BM, PBL, spleen (S) and thymus (T)were stained with the following rat anti-mouse antibodies: CD45-FITC(BM, PBL, T, S), CD4-PE (BM, PBL, T, S), CD8-APC (BM, PBL, T, S),Mac1/Gr1-PE (BM, PBL, S), B220-PerCP (BM, PBL, S), Sca-1-PE (BM), andc-kit-FITC (BM), and examined by flow cytometry. Percentages of theindicated hematopoietic cell subpopulations are expressed as mean % ± SDof total CD45+ cells (n = 3 per group). Cell population BM (%) PBL (%)Thymus (%) Spleen (%) CD45⁺ 92.3 ± 1.3 96.1 ± 2.7 98.7 ± 0.4 94.9 ± 1.8CD4⁺  4.9 ± 1.4 15.9 ± 0.8  6.5 ± 1.0 18.4 ± 2.7 CD8⁺  2.4 ± 0.2 11.7 ±0.7  2.9 ± 0.1 11.8 ± 1.9 CD4⁺/CD8⁺ 86.9 ± 0.9 B220⁺ 28.2 ± 2.7 55.7 ±2.9 51.3 ± 5.5 Mac1⁺/Gr1⁺ 79.7 ± 2.2 30.1 ± 4.0  9.9 ± 1.8 KLS (HSC) 3.3 ± 0.5

Taken together, these results suggested that up to two doses of 10 mg/kg6TG could be employed as an effective conditioning regimen that would bewell tolerated for up to 3 days prior to HSCT, with progressivelyincreasing myelotoxicity occurring over a period of 7 days.

Transplantation of HPRT-Deficient BM after 6TG Conditioning in HPRT-WtRecipients

Based on the schedule established above, we employed 6TG (10 mg/kg i.p.)as a conditioning regimen in HPRT-wt recipients (0045.1), with one doseadministered 48 hours prior (now designated Day −2, rather than Day 1)and one dose administered on the day of transplantation (designated Day0, rather than Day 3) per the schedule established above. Afterconditioning, HPRT-wt recipients were then transplanted with BM fromHPRT-deficient congenic donors (CD45.2),

At day 4, the marrow showed reduced cellularity with fewer earlyprogenitor cells and increased vascularity (FIG. 2B), and the overallcount of nucleated BM cells recovered from these 6TG-conditioned HPRT-wtmice transplanted with HPRT-deficient BM (3.8×10⁶±0.5×10⁶, n=4) wasstill significantly reduced (p<0.001) compared to that of the vehiclecontrol-treated HPRT-wt group (1.1×10⁷±0.2×10⁷, n=3). Flow cytometricanalyses of BM showed that at day 4 only 17.8%±4.4% of the total cellpopulation was derived from donor HPRT-deficient CD45.2+ hematopoieticcells.

Threshold for Myelotoxicity after Chronic Low-Dose 6TG Treatment

As our results above indicated that 6TG preconditioning alone may not besufficient to achieve high levels of engraftment, we next performed adose-finding study in non-transplanted mice given chronic treatment withlower doses of 6TG. HPRT-wt and HPRT-deficient mice (n=3 per group) wereinjected i.p. with vehicle alone, 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.5mg/kg, or 5.0 mg/kg 6TG every 3 days for up to 60 days. In HPRT-wt mice,the vehicle control group as well as the 0.25 mg/kg and 0.5 mg/kg 6TGgroups showed 100% survival over a period of 60 days, and histologicalexamination of BM in the 0.25 mg/kg and 0.5 mg/kg 6TG groups showednormal cellularity on Day 60 (FIG. 3).

In contrast, in the HPRT-wt 1.0 mg/kg 6TG group, deaths occurred on Day38 (13 mg/kg total dosage), Day 42 (14 mg/kg total dosage), and Day 51(17 mg/kg total dosage) (Table S1). At higher dosages, HPRT-wt micereceiving repeated injections of 2.5 mg/kg or 5 mg/kg 6TG consistentlyshowed progressive clinical signs of distress (inactivity, hunchedposture, lack of grooming, anorexia), anemia (pallor of extremities)and >10% weight loss, necessitating sacrifice per institutionalguidelines on Day 28 (2.5 mg/kg 6TG group; 22.5 mg/kg total dosage) andDay 22 (5.0 mg/kg 6TG group; 35 mg/kg total dosage), respectively.Histological examination of BM from the 1.0 mg/kg 6TG group showedseveral apoptotic figures and more blast cells than in the lower dosagegroups were observed, likely reflecting an initial activation responseto the injury (FIG. 3; Day 38). The higher dosage groups also showedsignificantly reduced cellularity and expansion of vascular structures,with the severity of lesions proportional to the cumulative dosage of6TG in each group. Mice treated with repeated doses of 2.5 mg/kg 6TGshowed reduced cellularity and widespread apoptosis (Day 28). At thehighest chronic dosage of 5 mg/kg, BM was markedly depleted, with mostof the surviving cells being of myeloid lineage (Day 22).

In contrast to the HPRT-wt mice, all HPRT-deficient mice survived forthe duration of the experiment (60 days), independent of the injected6TG dosage (up to 105 mg/kg maximum total dose, administered accordingto the same dosing schedules as above) (Table S1). No significant BMpathology was observed in any HPRT-deficient mice, regardless of 6TGdosage, at the terminal time point of the experiment on Day 60 (FIG. 3).No significant treatment-related abnormalities were observed in anyother tissues examined, including heart, lung, liver, pancreas, kidney,and spleen. Thus, HPRT-deficient mice showed no toxic effects of chronic6TG treatment at the 1.0 mg/kg, 2.5 mg/kg, and 5.0 mg/kg doses thatcaused lethal myelotoxicity in HPRT-wt mice.

Combined 6TG Conditioning and In Vivo Chemoselection Achieves Consistentand Highly Efficient Engraftment of HPRT-Deficient BM

Based on the above dose-finding study, we then asked whether 6TGpreconditioning combined with continued administration of lower doses of6TG, beginning immediately after transplantation of HPRT-deficient BM inHPRT-wt recipients, could achieve further chemoselective amplificationof engrafted donor cells. Accordingly, female HPRT-wt mice werepreconditioned with 6TG (10 mg/kg i.p.), with one dose administered 48hours prior (Day −2) and one dose administered on the day oftransplantation (Day 0) per the conditioning schedule establishedpreviously. The HPRT-wt female recipients were then transplanted withHPRT-deficient male BM, and based on the chronic myelotoxicity resultsabove, the recipients were further treated with repeated doses of 2.5mg/kg 6TG every 3 days for 2 weeks (30 mg/kg total dosage) or 4 weeks(42.5 mg/kg), or repeated doses of 5.0 mg/kg 6TG every 3 days for 2weeks (40 mg/kg total dosage) or 4 weeks (65 mg/kg total dosage).Analysis was performed immediately after the in vivo chemoselectionperiod at 2 weeks or 4 weeks, respectively (FIG. 4).

As expected, the combined 6TG conditioning, HSCT, and 6TG in vivochemoselection procedures were well-tolerated, and no signs of distresswere observed. In all 6TG-treated groups, 100% of the transplantedanimals survived (2 week treatment: n=3 and 4 week treatment: n=8). Bodyweights decreased initially during the first week after transplantationin the 6TG-treated animals, but stabilized and all animals regainednormal weight thereafter. Histopathological analysis showed that overallcellularity and hematopoiesis in the transplanted animals wereindistinguishable from the untreated HPRT-wt control, regardless of 6TGchemoselection dosage or duration (FIG. 4).

Chromosome XY-FISH [25, 26] showed that in the groups receiving 2 weeksof 6TG chemoselection, the BM at that time point was already highlyreconstituted with donor-derived marrow at levels of 89.3%±1.7 (2.5mg/kg group) and 95.5%±1.2 (5 mg/kg group). The percentages ofdonor-derived peripheral blood leukocytes (PBL) at 2 weeks were13.0%±4.6 (2.5 mg/kg group) and 12.7%±2.9 (5.0 mg/kg group),respectively (Table 4). When in vivo chemoselection was continued for upto 4 weeks after HSCT, the percentage of donor-derived BM cells wasagain found to be extremely high in both the 2.5 mg/kg group (95.3%±0.9)and the 5.0 mg/kg group (96.7%±1.2). Notably, the percentage ofdonor-derived PBL was significantly higher in the 5.0 mg/kg group(39.7%±3) in comparison to the 2.5 mg/kg group (29.9%±1.9) at 4 weeks(p<0.005), as well as significantly higher (p<0.0002) than in the groupsreceiving 2 weeks of chemoselection at either dose (Table 4). Thus,preconditioning with 10 mg/kg 6TG on Day −2 and Day 0, combined withon-going in vivo chemoselection with 5 mg/kg 6TG every 3 days for 4weeks, yielded maximal levels of BM engraftment by HPRT-deficient donorcells as well as the highest levels of donor-derived PBL; this regimenwas employed in further studies.

TABLE 4 Survival and engraftment after combined 6TG conditioning and invivo chemoselection. Treatment schedules for in vivo chemoselection areas indicated: In vivo chemoselection with 2.5 mg/kg 6TG for 2 weeks, 5.0mg/kg 6TG for 2 weeks, 2.5 mg/kg 6TG for 4 weeks or 5.0 mg/kg for 4weeks. All treatment groups showed 100% survival. Engraftment ofHPRT-deficient male hematopoietic cells in HPRT-wt female recipient BMand PBL was determined by chromosome XY-FISH (mean % ± SD). EngraftmentTreatment Survival (%) BM (%) PBL (%) 6TG 2.5 mg/kg × 100 89.3 ± 1.713.0 ± 4.6 2 weeks (n = 3) 6TG 5.0 mg/kg × 100 95.5 ± 1.2 12.7 ± 2.9 2weeks (n = 3) 6TG 2.5 mg/kg × 100 95.3 ± 0.9 29.9 ± 1.9 4 weeks (n = 3)6TG 5.0 mg/kg × 100 96.7 ± 1.2 39.7 ± 3.0 4 weeks (n = 8)

Combined 6TG Conditioning and In Vivo Chemoselection Results inLong-Term Reconstitution of HPRT-Deficient BM

The durability of engraftment by HPRT-deficient donor BM using thecombined 6TG conditioning and in vivo chemoselection regimen establishedabove was examined 4 months, 7 months, or 12 months aftertransplantation (i.e., 3 months, 6 months, and 11 months after the endof the 4-week in vivo chemoselection period, respectively). Alltransplanted animals (4 months: n=8; 7 months: n=6; 12 months: n=5)remained alive and well, showing no signs of any morbidity ordiscomfort, at all time points examined. Gross pathological andhistological examination of these animals revealed no significantabnormalities.

Multi-lineage reconstitution of lymphohematopoiesis by donor-derivedprogenitor cells was evaluated 4 months after HSCT with combined 6TGpreconditioning and in vivo chemoselection, i.e., 3 months after the endof the 4-week chemoselection period. Immunophenotyping of BM, PBL,thymus, and spleen was performed in a congeneic C045.1/0045.2 transplantsetting using HPRT-deficient CD45.2 mice as donors and HPRT-wt CD45.1mice as recipients (n=5). All hematopoietic tissues showed highengraftment of CD45.2+ donor cells at levels exceeding 75% of total bonemarrow (Table 5). Immunophenotyping of the donor-derived CD45.2⁺population showed that the relative percentages of T cells (CD4/CD8),B220+ cells, and macrophages/granulocytes (Mac-1/Gr1) were comparable tothose of the treatment-naïve controls (Tables 2 and 3).

TABLE 5 Immunophenotypic analysis of hematopoietic tissues 4 monthspost-transplantation using 6TG conditioning and in vivo chemoselectionregimen in a congeneic CD45.1/CD45.2 transplant model. 6TG conditioningand in vivo chemoselection was performed as described in text. RecipientBM and PBL were stained with the following rat anti-mouse antibodies:CD45.2-FITC (BM, PBL, T, S), CD4-PE (BM, PBL, T, S), CD8-APC (BM, PBL,T, S), Mac1/Gr1-PE (BM, PBL), B220-PerCP (BM, PBL, S), and examined byflow cytometry. Percentages of the indicated hematopoietic cellsubpopulations are expressed as mean % ± SD of total CD45.2+ cells (n =5 per group). Cell population BM (%) PBL (%) Thymus (%) Spleen (%)CD45.2⁺ 76.1 ± 9.3 73.8 ± 5.2 89.7 ± 8.3 65.9 ± 5.2 CD4⁺  3.2 ± 1.3 17.0± 3.0 10.4 ± 2.8 22.6 ± 3.6 CD8⁺  3.0 ± 1.3 12.7 ± 0.9  4.3 ± 0.8 14.9 ±2.1 CD4⁺/CD8⁺ 79.3 ± 4.2 B220⁺ 29.3 ± 6.3 45.4 ± 3.8 58.2 ± 8.6Mac1⁺/Gr1⁺ 80.68 ± 6.1  28.2 ± 3.3 14.1 ± 1.6

Engraftment levels were also evaluated by chromosome XY-FISH at thepost-transplant 4-month, 7-month, and 12-month time points. Stablelong-term reconstitution by donor-derived BM at high levels was observedat all time points, with engraftment levels of 97.7%±0.5% (4 months),94.7%±1.9% (7 months), and 93.0%±0.8% (12 months), respectively, aftertransplantation (FIG. 5). Furthermore, the percentage of donor-derivedPBL was significantly increased (67.4%±10.6% at 4 months, p=0.01;73.3%±7.9% at 7 months, p<0.01; 73.0%±10.7% at 12 months, p<0.01)compared to that immediately following the 4-week 6TG selection period(39.7%±3.0%).

The relative percentages of CD4+ and CD8+ cells, B220+ cells, andMac-1+/Gr1+ cells, as well as KLS (lin⁻/sca-1⁺/c-kit⁺) HSC, weredetermined in BM by immunophenotyping at 4 months, 7 months, and 12months (Table 6), and compared to treatment-naïve female HPRT-wt (Table2) and treatment-naïve male HPRT-deficient (Table 3) control mice. Atall three time points, the relative percentage of each cell populationwas comparable to that in the controls, although KLS cells were elevatedat 4 months and 7 months, and within the normal range at 12 months afterHSCT. Thus, HPRT-deficient donor-derived marrow was able to achievelong-term reconstitution of normal hematopoiesis for at least 12 monthspost-transplantation.

TABLE 6 Immunophenotypic analysis of BM at 4 months, 7 months, and 12months post-transplantation with HPRT-deficient BM using 6TGconditioning and in vivo chemoselection regimen. At the indicated timepoints after HSCT with 6TG conditioning and in vivo chemoselection,recipient BM cells were stained with the following rat anti-mouseantibodies: CD45-FITC, CD4-PE, CD8-APC, Mac1/Gr1-PE, B220-PerCP,Sca-1-PE, and c-kit-FITC, and examined by flow cytometry. Percentages ofthe indicated hematopoietic cell subpopulations are expressed as mean %± SD of total CD45+ cells. Cell 4 months 7 months 12 months populationBM (%) BM (%) BM (%) CD45⁺ 91.6 ± 1.9  94.3 ± 2.1  82.5 ± 4.9  CD4⁺ 2.5± 0.6 3.8 ± 0.2 2.8 ± 2.7 CD8⁺ 1.9 ± 0.1 2.2 ± 0.4 1.8 ± 0.6 B220⁺ 51.8± 2.4  32.9 ± 3.8  11.5 ± 2.8  Mac1⁺/Gr1⁺ 77.0 ± 13.0 80.8 ± 2.3  72.3 ±5.4  KLS (HSC) 7.8 ± 1.0 7.0 ± 1.1 1.8 ± 0.6

Hematopoietic Reconstitution of Secondary Recipients after SerialTransplantation of HPRT-Deficient Donor BM with Combined 6TGConditioning and Chemoselection

To further evaluate whether the optimized regimen combining 6TGconditioning with chemoselection selects for long-term repopulatingHSCs, we next transplanted BM from primary recipients at 7 monthspost-transplantation into secondary recipients [27] using the sameregimen. The secondary transplant recipients were then maintained for 3months after the end of their 4-week course of 6TG in vivochemoselection (Supplementary Figure S3). After 6TG conditioning andchemoselection, HPRT-deficient male donor cells that had engraftedprimary recipients were able to serially repopulate secondary femalerecipients at high levels, (95.5%±1.1%, as determined by XY-FISH).Immunophenotypic analysis revealed that the percentages of all cellpopulations examined in BM and PBL of secondary recipients (Table 7)were again comparable to those of treatment-naïve controls (Tables 2 and3).

TABLE 7 Immunophenotypic analysis of hematopoietic cells in secondaryrecipients after serial transplantation of HPRT-deficient BM with 6TGconditioning and in vivo chemoselection. Serial transplantation with 6TGconditioning and in vivo chemoselection regimen was performed asdescribed in FIG. S3. Secondary recipient BM and PBL were stained withthe following rat anti-mouse antibodies: CD45-FITC (BM, PBL), CD4-PE(BM, PBL), CD8-APC (BM, PBL), Mac1/Gr1-PE (BM, PBL), and B220-PerCP (BM,PBL), and examined by flow cytometry. Percentages of the indicatedhematopoietic cell subpopulations are expressed as mean % ± SD of totalCD45+ cells (n = 6 per group). Cell population BM (%) PBL (%) CD45⁺ 84.6± 4.7 95.8 ± 1.4  CD4⁺  4.1 ± 1.0 7.0 ± 3.3 CD8⁺  2.4 ± 0.8 7.0 ± 3.2B220⁺ 19.1 ± 2.9 33.3 ± 12.3 Mac1⁺/Gr1⁺ 78.3 ± 4.7 31.6 ± 11.7

We have developed an optimized regimen that employs 6TG as a singleagent for pre-transplant conditioning as well as continued posttransplant in vivo chemoselection of HPRT-deficient donor HSC. Thiscombined 6TG conditioning and chemoselection strategy achieved efficientHSC engraftment with low overall toxicity, through a progressive andsimultaneous replacement process, in which recipient hosts showed littleif any distress and 100% survival, while their BM was rapidly and almostcompletely replaced by HPRT-deficient donor cells, consistentlyachieving ˜95% engraftment by XY-FISH, and >75% by CD45.2immunophenotyping. The percentage of donor-derived PBL increasedsignificantly over time (4 and 7 months post-transplantation) comparedto that immediately following the 4-week 6TG selection period. Residualrecipient cells in PBL likely reflect a lower turnover of non-dividingmature cells in PBL. Stable long-term reconstitution of the BM wasachieved in both primary and secondary recipients. Immunophenotypinganalysis of BM, PBL, spleen, and thymus showed that, after long-termreconstitution, hematopoietic differentiation was unaffected by 6TG invivo chemoselection.

These results also confirm our previous observation that, at appropriateconcentrations, 6TG appears to induce selective myelotoxicity withoutany adverse effects on extra-hematopoietic tissues. HPRT is expressed atlow levels in all somatic cells [30], and inherited loss of HPRT givesrise to Lesch-Nyhan syndrome [31] which manifests as severe mentalretardation and behavioral abnormalities, as higher levels of HPRTexpression and activity have been found in the central nervous system,particularly during neural development [32]. However, since fullydifferentiated neurons do not undergo replication, these higher levelsdo not translate into higher 6TG neurotoxicity in mature adults.

In previous work by Porter and DeGregori [22], total body irradiation of4.5 Gy was used to achieve myeloablation, and 6TG was employed only forchemoselection at later time periods. In contrast, in our regimen, 6TGas a single agent fulfills the dual role of conditioning (cytoreductionof host BM) and chemoselective drug (amplification of donor BM). It isknown that 6TG toxicity requires two rounds of DNA replication to resultin apoptosis [33] and therefore shows a delayed effect. Our results alsoindicated a dose- and time-dependent myelotoxic effect of 6TG. Thus,conditioning with 10 mg/kg 6TG prior to transplantation may improve theoutcome of 6TG in vivo chemoselection by compensating for the delay in6TG myelotoxicity and providing an adequate niche for HSC at the time oftransplantation.

In addition, Porter et al. employed 6TG at much lower doses ranging from0.25-2 mg/kg over short periods of time, starting more than a monthafter transplantation, resulting in variable engraftment levels rangingfrom 5-50%. In our current study, we have confirmed that micetransplanted with Hprt-deficient BM can tolerate 6TG doses 5- to 40-foldhigher, with administration of the chemoselective drug startedimmediately post-transplant, and continued over considerably longerselection periods.

In order to translate the use of the 6TG in vivo chemoselection strategyinto a clinically feasible approach, it is necessary to develop methodsto genetically engineer normal HSC to render them HPRT-deficient andthus 6TG-resistant. Furthermore, it should be emphasized that ourcurrent study was limited to employing this strategy for bone marrowtransplantation in syngeneic mice, which models the situation in theautologous transplant setting. Whether this strategy will be equallyuseful in the allogeneic setting remains to be seen, asreduced-intensity conditioning regimens are being used routinely tocircumvent the toxicity of traditional myeloablative regimens. Potentialissues that may need to be addressed include the possibility ofspontaneous 6TG resistance arising in leukemic cells after allogeneictransplantation for hematopoietic malignancies, and possibleexacerbation of graft-vs.-host disease when allogeneic donor cells areselectively amplified in vivo.

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Example 2: Creation of HPRT-Deficient Cells

This example is directed at translational application to ex vivo genetherapy in the autologous setting, employing both third-generationlentiviral vectors expressing different HPRT-targeted shRNA candidatesequences, as well as nucleofection of zinc finger nucleases (ZFN)targeting HPRT. The latter approach is advantageous as only transientexpression of the ZFN construct is needed to achieve permanent knockoutof the target gene, thereby mitigating the potential for insertionalmutagenesis as a result of the genetic engineering procedure. In thiscontext, this strategy is unique in imparting a selective advantage totransplanted cells through an enzyme deficiency, rather than inserting anew transgene to achieve chemoresistance. HPRT-targeted shRNA wassuccessful in down-regulating HPRT to undetectable levels. In addition,HPRT has been successfully knocked out using ZFN.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of radiation-free hematopoietic stem cell (HSC)transplantation, the method comprising: (a) engrafting into apre-conditioned mammalian subject hypoxanthine-guaninephosphoribosyltransferase (HPRT)-deficient donor HSCs; and (b)immediately administering to the subject about 1 to 5 mg/kg of a purinebase analog selected from 6-thioguanine (6TG), 6-mercaptopurine (6-MP),or azathiopurine (AZA) every two to four days for two to eight weeks;wherein the method is performed in the absence of pre-conditioning viaradiation.
 2. The method of claim 1, wherein the HSCs have beengenetically modified.
 3. The method of claim 2, wherein the HSCs havebeen genetically modified to correct a hereditary genetic defect, toalter drug sensitivity of normal bone marrow to cytotoxic drugs, toconfer resistance to infectious microorganisms that affectlymphohematopoietic cells, to replace or re-set the endogenous immunesystem, or to combat lymphohematopoietic malignancies throughreplacement of endogenous bone marrow and induction of agraft-vs.-leukemia/lymphoma effect.
 4. The method of claim 1, whereinthe subject is human.
 5. The method of claim 1, wherein the subject hasa hereditary or genetic disorder.
 6. The method of claim 1, wherein thesubject has an acquired disease affecting lymphohematopoietic cells. 7.The method of claim 6, wherein the disease is human immunodeficiencyvirus (HIV) infection or acquired immune deficiency syndrome (AIDS). 8.The method of claim 6, wherein the disease is a lymphohematopoieticmalignancy.
 9. The method of claim 1, wherein the subject has a diseaseor disorder of hematopoietic or thrombopoietic or lymphopoietic system.10. The method of claim 9, wherein the disease or disorder of thehematopoietic system is a hemoglobinopathy.
 11. The method of claim 1,wherein the method further comprises bone marrow transplantation. 12.The method of claim 1, wherein the purine base analog is 6TG.
 13. Themethod of claim 1, wherein the total 6TG dosage administered to thesubject in the administering of step (b) does not exceed 105 mg.
 14. Themethod of claim 1, wherein the total 6TG dosage administered to thesubject in the administering of step (b) does not exceed 75 mg.
 15. Themethod of claim 1, wherein the administering of step (b) is performedevery 3 days and for not more than four weeks following the engraftingstep.
 16. The method of claim 1, wherein the subject exhibits over 75%genetically modified hematopoietic cells.
 17. The method of claim 1,wherein the subject exhibits over 95% genetically modified hematopoieticcells.
 18. The method of claim 1, wherein the HPRT-deficient HSCs to betransplanted have been rendered HPRT-deficient via introduction ofsequences encoding zinc finger nucleases (ZFNs), transcriptionalactivator-like effector nucleases (TALENs), small fragment homologousrecombination (SFHR) template strands, inhibitory RNAs (siRNAs) ormicroRNAs (miRNAs), antisense RNAs, trans-splicing RNAs, ribozymes,intracellular antibodies, or dominant-negative or competitive inhibitorproteins.
 19. The method of claim 1, wherein the HPRT-deficient HSCs tobe transplanted have been rendered HPRT-deficient via introduction ofsequences encoding zinc finger nucleases (ZFNs).
 20. The method of claim1, wherein the HPRT-deficient HSCs to be transplanted have been renderedHPRT-deficient via introduction of sequences encoding transcriptionalactivator-like effector nucleases (TALENs).
 21. The method of claim 1,wherein the HPRT-deficient HSCs to be transplanted have been renderedHPRT-deficient via introduction of sequences encoding inhibitory RNAs(siRNAs).
 22. The method of claim 1, wherein the transplanted HSCs areautologous or syngeneic.
 23. The method of claim 1, wherein thetransplanted HSCs are allogeneic.
 24. The method of claim 1, wherein thesubject is not treated with myeloablative radiation.
 25. A method oftreating symptoms of a disease or disorder in a mammalian subject, themethod comprising: (a) administering a pre-conditioning treatment to thesubject; (b) engrafting into the subject hypoxanthine-guaninephosphoribosyltransferase (HPRT)-deficient donor HSCs; and (c)immediately administering to the subject about 1 to 5 mg/kg of a purinebase analog selected from 6-thioguanine (6TG), 6-mercaptopurine (6-MP),or azathiopurine (AZA) every two to four days for two to eight weeks;wherein the method is performed in the absence of pre-conditioning viaradiation.
 26. The method of claim 25, wherein the HSCs have beengenetically modified to correct a hereditary genetic defect, to alterdrug sensitivity of normal bone marrow to cytotoxic drugs, to conferresistance to infectious microorganisms that affect lymphohematopoieticcells, to replace or re-set the endogenous immune system, or to combatlymphohematopoietic malignancies through replacement of endogenous bonemarrow and induction of a graft-vs.-leukemia/lymphoma effect.
 27. Themethod of claim 25, wherein the subject has human immunodeficiency virus(HIV) infection or acquired immune deficiency syndrome (AIDS).
 28. Themethod of claim 25, wherein the subject has a lymphohematopoieticmalignancy.
 29. The method of claim 25, wherein the subject has adisease or disorder of hematopoietic or thrombopoietic or lymphopoieticsystem.
 30. The method of claim 29, wherein the disease or disorder ofthe hematopoietic system is a hemoglobinopathy.
 31. The method of claim25, wherein the method further comprises bone marrow or hematopoieticstem cell transplantation.
 32. The method of claim 25, wherein thesubject is human.
 33. The method of claim 25, wherein the purine baseanalog is 6TG.
 34. The method of claim 25, wherein the administering ofstep (c) is performed every 3 days and for not more than four weeksfollowing the engrafting step.
 35. The method of claim 25, wherein thesubject exhibits over 75% genetically modified hematopoietic cells. 36.The method of claim 25, wherein the subject exhibits over 95%genetically modified hematopoietic cells.
 37. The method of claim 25,wherein the HPRT-deficient HSCs to be transplanted have been renderedHPRT-deficient via introduction of sequences encoding zinc fingernucleases (ZFNs), transcriptional activator-like effector nucleases(TALENs), small fragment homologous recombination (SFHR) templatestrands, inhibitory RNAs (siRNAs) or microRNAs (miRNAs), antisense RNAs,trans-splicing RNAs, ribozymes, intracellular antibodies, ordominant-negative or competitive inhibitor proteins.
 38. The method ofclaim 25, wherein the HPRT-deficient HSCs to be transplanted have beenrendered HPRT-deficient via introduction of sequences encoding zincfinger nucleases (ZFNs).
 39. The method of claim 25, wherein theHPRT-deficient HSCs to be transplanted have been rendered HPRT-deficientvia introduction of sequences encoding transcriptional activator-likeeffector nucleases (TALENs).
 40. The method of claim 25, wherein theHPRT-deficient HSCs to be transplanted have been rendered HPRT-deficientvia introduction of sequences encoding inhibitory RNAs (siRNAs).
 41. Themethod of claim 25, wherein the transplanted HSCs are autologous orsyngeneic.
 42. The method of claim 25, wherein the transplanted HSCs areallogeneic.
 43. The method of claim 25, wherein the subject is nottreated with myeloablative radiation.